McShea Hanon Solomon, Viens Robb A, Olagunju Babatunde O, Giner José-Luis, Welander Paula V
Department of Earth System Science, Stanford University, Stanford, CA, USA.
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
bioRxiv. 2025 Aug 2:2024.10.28.620730. doi: 10.1101/2024.10.28.620730.
Cyclic terpenoids are a class of lipid compounds containing immense structural and functional diversity, with many cyclic triterpenoids acting as regulators of the physical properties and spatial organization of lipid membranes. Cyclic terpenoids are also readily preserved as terpane fossils, such as steranes and hopanes, forming a rich record of the evolution of life on Earth. Formation of the multiple ring structure of all cyclic terpenoids is catalyzed by terpenoid cyclase enzymes, among which are whole clades of proteins - many from environmental metagenomes and uncultured organisms - whose substrates and products are completely unknown. We investigate the function of these divergent cyclases through biochemical assays, and the evolutionary processes that produced them by testing and applying a variety of evolutionary models. We find deep divergence between the diterpenoid cyclases and triterpenoid cyclases, with other clades branching between the two, rooting the triterpenoid cyclase subtree, between squalene-hopene cyclases and sterol cyclases. Through a simple test of evolutionary rate shifts, we find an elevated evolutionary rate in the enzyme active site on the squalene-hopene cyclase stem, potentially indicative of positive selection. Finally, by testing the activity of divergent cyclases for a variety of substrates, we find a group of early-branching sterol cyclases from bacteria that synthesize arborinols, two of which produce the molecular precursor to a Permian "orphan biomarker." Together, our data present an evolutionary framework for triterpenoid cyclases that can inform both the biochemical potential of these proteins and their products' occurrence in the geological record.
环状萜类化合物是一类具有巨大结构和功能多样性的脂质化合物，许多环状三萜类化合物作为脂质膜物理性质和空间组织的调节剂。环状萜类化合物也很容易以萜烷化石的形式保存下来，如甾烷和藿烷，形成了地球上生命进化的丰富记录。所有环状萜类化合物的多环结构形成均由萜类环化酶催化，其中有一整个蛋白质分支——许多来自环境宏基因组和未培养生物——其底物和产物完全未知。我们通过生化分析研究这些不同环化酶的功能，并通过测试和应用各种进化模型来研究产生它们的进化过程。我们发现二萜环化酶和三萜环化酶之间存在深度分化，其他分支在两者之间，三萜环化酶子树的根部位于鲨烯 - 藿烯环化酶和甾醇环化酶之间。通过对进化速率变化的简单测试，我们发现在鲨烯 - 藿烯环化酶主干上的酶活性位点进化速率升高，这可能表明存在正选择。最后，通过测试不同环化酶对各种底物的活性，我们发现了一组来自细菌的早期分支甾醇环化酶，它们能合成乔木醇，其中两种能产生二叠纪“孤儿生物标志物”的分子前体。总之，我们的数据为三萜环化酶提供了一个进化框架，这可以为这些蛋白质的生化潜力及其产物在地质记录中的出现提供信息。
